Introduction Usage Parameters Output

Introduction

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

  • NumorphIntensity - Mesures intensity parameters for illumination correction
  • NumorphAlign - Performs 2D rigid or 3D non-linear channel alignment
  • NumorphStitch - Performs 2D iterative stitching of image tiles
  • NumorphResample - Generate downsamppled images
  • NumorphRegister - Performs registartion to the Allen Reference Atlas (ARA)
  • [Mat2JSON] (#mat3json) - Converts .matfiles to JSON
  • Pipeline information - Report metrics generated during the workflow execution

NumorphIntensity

Output files
  • Sample_id/intesnity/results
    • NM_variables.mat: Contains input and calculated parameters
    • samples/
      • [channel]_x_y_z.tif: Image comparing raw and adjusted image tiles for respective channel. Example for each tile of the middel z slice
      • flatfield_*.png: Flatfield correction heatmap for each channel
      • tile_adj_*.png: Heatmap of tile positions displaying illumination correction factor for each tile
      • y_adj_*.png: Intensity correction factors along the Y-axis(using intensity profiles specifically for the LaVision Ultramicroscope II)
    • variables/
      • adj_params.mat: Correction parameters
      • thresholds.mat: Intensity thresholds per channel
      • path_table.mat: Image information table

NumorphIntensity calculates intensity thresholds and performs intensity adjustments in the y dimension and between image tile stacks on raw images. The process includes the MATLAB implementation of BaSiC for shading correction.

NumorphAlign

Output files
  • Sample_id/alignment/results
    • NM_variables.mat: Contains input and calculated parameters
    • samples/
      • Sample_id_*_full.tif: Aligned image for each channel
    • variables/
      • alignment_table.mat: Alignment table
      • z_displacement_align.mat: Z displacement matrix for given channels to the reference
      • path_table.mat: Image information table

NumorphAlign performs rigid or non-rigind alignemnt of channels to a reference (nuclei) channel and determines z displacement per tile for each channel.

NumorphStitch

Output files
  • Sample_id/stitching/results
    • NM_variables.mat: Contains input and calculated parameters
    • stitched/
      • Sample_id_*_stitched.tif: Stitched images per z slice
    • variables/
      • adj_params.mat: Correction parameters
      • thresholds.mat: Intensity thresholds per channel
      • path_table.mat: Image information table
      • stitch_tforms.mat: Stitching information
      • adjusted_z.mat: Adjusted z positions for each tile
      • z_disp_matrix.mat: Z displacement for adjecent tiles
      • z_displacement_align.mat: Z displacement matrix for given channels to the reference

NumorphStitch performs 2D iterative stitching.

NumorphResample

Output files
  • Sample_id/resampled/results
    • NM_variables.mat: Contains input and calculated parameters
    • Sample_id_*.nii: Downsampled image

NumorphResample downsamples the image resolition to macht the Allen Reference Atlas resolution befor registration.

NumorphRegister

Output files
  • Sample_id/results - NM_variables.mat: Contains input and calculated parameters - registered/*_MOV_*.nii: Moving image from registration - registered/*_REF_*.nii: Reference imgae from registration - variables/reg_params.mat: Registration parameters

NumorphRegister performs image registration to the Allen Reference Atlas.

Mat2JSON

Output files

  • process/

    • *.json/*.csv: Converted mat file

Mat2JSON converts a given .matfile into a CSV if the data is stored as a table datastructure or a JSON for other nested datastructures.

Pipeline information

Output files
  • pipeline_info/
    • Reports generated by Nextflow: execution_report.html, execution_timeline.html, execution_trace.txt and pipeline_dag.dot/pipeline_dag.svg.
    • Reports generated by the pipeline: pipeline_report.html, pipeline_report.txt and software_versions.yml. The pipeline_report* files will only be present if the --email / --email_on_fail parameter’s are used when running the pipeline.
    • Reformatted samplesheet files used as input to the pipeline: samplesheet.valid.csv.
    • Parameters used by the pipeline run: params.json.

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.
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